1. Field of the Invention
The present invention relates to a novel inhibitor of TNFR-1 and CD-95 induced apoptosis. More specifically, isolated nucleic acid molecules are provided encoding a human I-FLICE (Inhibitor of FLICE (FADD-like ICE)) polynucleotides. I-FLICE polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of I-FLICE activity. Also provided are therapeutic methods for treating diseases and disorders associated with apoptosis.
2. Related Art
The cell death machinery is conserved throughout evolution and is composed of activators, inhibitors, and effectors (Chinnaiyan, A. M. and Dixit, V. M., Curr. Biol. 6:555-562 (1996)). The effector arm of the cell death pathway is composed of a rapidly growing family of cysteine aspartate-specific proteases termed caspases (Alnemri, E. S., et al., Cell 87:171 (1996)). As implied by the name, these cysteine proteases cleave substrates following an aspartate residue (Alnemri, E. S., et al., Cell 87:171 (1996); Walker, N. P., et al., Cell 78:343-352 (1994)). Caspases are normally present as single polypeptide zymogens and contain an amino-terminal prodomain, and large and small catalytic subunits (Wilson, K. P., et al., Nature 370:270-274 (1994); Rotonda, J., et al.; Nat. Struct. Biol. 3:619-625 (1996); Fraser, A. and Evan, G., Cell 85:781-784 (1996)). The two chain active enzyme (composed of the large and small subunits) is obtained following proteolytic processing at internal Asp residues (Wilson, K. P., et al., Nature 370:270-274 (1994); Rotonda, J., et al., Nat. Struct. Biol. 3:619-625 (1996); Fraser, A. and Evan, G., Cell 85:781-784 (1996)). As such, caspases are capable of activating each other in a manner analogous to zymogen activation that is observed in the coagulation cascade (Boldin, M. P., et al., Cell 85:805-815 (1996)). The identification of FLICE and Mch4/FLICE2 as receptor associated caspases suggested a surprisingly direct mechanism for activation of the death pathway by the cytotoxic receptors CD-95 and TNFR-1 (Boldin, M. P., et al., Cell 85:805-815 (1996); Muzio, M., et al., Cell 85:817-827 (1996); Vincenz, C. and Dixit, V. M., J. Biol. Chem. 272:6578-6583 (1997); Chinnaiyan, A. M., et al., Cell 81:505-512 (1995)). Upon activation, both receptors use their death domains to bind the corresponding domain in the adaptor molecule FADD (Fas-associated death domain protein) (Muzio, M., et al., Cell 85:817-827 (1996); Vincenz, C. and Dixit, V. M., J. Biol. Chem. 272:6578-6583 (1997); Chinnaiyan, A. M., et al., Cell 81:505-512 (1995)). Dominant negative versions of FADD that lack the N-terminal segment but still retain the death domain potently inhibit both CD-95 and TNFR-1 induced apoptosis (Chinnaiyan, A. M., et al., J. Biol. Chem. 271:4961-4965 (1996); Muzio, M., et al., J. Biol. Chem. 272:2952-2956 (1997)). Given the importance of the N-terminal segment in engaging the death pathway, it has been termed the death effector domain (DED) (Chinnaiyan, A. M., et al., J. Biol. Chem. 271:4961-4965 (1996)).
Remarkably, the DED is present within the prodomain of FLICE and Mch4/FLICE2 and mutagenesis studies suggest that a homophilic interaction between the DED of FADD and the corresponding domain in FLICE or Mch4/FLICE2 is responsible for the recruitment of these proteases to the CD-95 and TNFR-1 signalling complexes (Muzio, M., et al., Cell 85:817-827 (1996); Vincenz, C. and Dixit, V. M., J. Biol. Chem. 272:6578-6583 (1997); Chinnaiyan, A. M., et al., Cell 81:505-512 (1995); Chinnaiyan, A. M., et al., J. Biol. Chem. 271:4961-4965 (1996)). Taken together, these data are consistent with FLICE and Mch4/FLICE2 being apical enzymes that initiate precipitous proteolytic processing of downstream caspases resulting in apoptosis (Boldin, M. P., et al., Cell 85:805-815 (1996); Srinivasula, S. M., et al., PNAS 93:14486-14491 (1996); Fernandes-Alnemri, T., et al., PNAS 93:7464-7469 (1996); Henkart, P. A., Immunity 4:195-201 (1996)). A number of viral gene products antagonize CD-95 and TNFR-1 mediated killing as a means to persist in the infected host (Shen, Y. and Shenk, T. S., Current Opinion in Genetics and Development 5:105-111 (1995)). The poxvirus encoded serpin CrmA and baculovirus gene product p35 are direct caspase inhibitors (Walker, N. P., et al., Cell 78:343-352 (1994)). In contrast, the molluscum contagiosum virus protein MC159 and the equine herpes virus protein E8 encode DED-containing decoy molecules that bind to either FADD (MC159) or FLICE (E8) and disrupt assembly of the receptor signalling complex, thereby abrogating the death signal (Hu, S., et al., J. Biol. Chem. 272:9621-9624 (1997); Bertin, J., et al., PNAS 94:1172-1176 (1997); Thome, M., et al., Nature 386:527-521 (1997)). The existence of these viral inhibitors has raised the question of whether functionally equivalent molecules are encoded in the mammalian genome.
There is a need for factors, such as the polypeptides of the present invention, that are useful for inhibiting apoptosis for therapeutic purposes, for example, in the treatment of Alzheimer""s disease, Parkinson""s disease, rheumatoid arthritis, septic shock, sepsis, stroke, CNS inflammation, osteoporosis, ischemia, reperfusion injury, cell death associated with cardiovascular disease, polycystic kidney disease, apoptosis of endothelial cells in cardiovascular disease, degenerative liver disease, MS and head injury damage. There is a need, therefore, for the identification and characterization of such factors that are inhibitors of apoptosis, such as the I-FLICE-1 and I-FLICE-2 polypeptides of the present invention, which can play a role in preventing, ameliorating or correcting the diseases and disorders associated with apoptosis.
The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the I-FLICE-1 polypeptide having the amino acid sequence shown in SEQ ID NO:2 or the amino acid sequence encoded by the cDNA clone deposited in a bacterial host as ATCC Deposit Number, 209041 on May 15, 1997. The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the I-FLICE-2 polypeptide having the amino acid sequence shown in SEQ ID NO:6 or the amino acid sequence encoded by the cDNA clone deposited in a bacterial host as ATCC Deposit Number 209038 on May 15, 1997.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of I-FLICE-1 or I-FLICE-2 polypeptides or peptides by recombinant techniques.
The invention further provides an isolated I-FLICE-1 or I-FLICE-2 polypeptides having an amino acid sequence encoded by the polynucleotides described herein.
The invention further provides a diagnostic method useful during diagnosis or prognosis of a disease states resulting from aberrant cell proliferation due to alterations in I-FLICE-1 or I-FLICE-2 expression.
The present invention also provides a screening method for determining whether a candidate agonist or antagonist is capable of enhancing or inhibiting a cellular activity of either an I-FLICE-1 or I-FLICE-2 polypeptide. The method involves contacting cells which express one or both of the I-FLICE-1 or I-FLICE-2 polypeptides with a candidate compound, assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed in absence of the candidate compound, whereby an increased cellular response over the standard indicates that the candidate compound is an agonist of the polypeptide activity and a decreased cellular response compared to the standard indicates that the candidate compound is an antagonist of the activity.
An additional aspect of the invention is related to a method for treating an individual in need of an increased level of I-FLICE-1 or I-FLICE-2 activity in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an isolated I-FLICE-1 or I-FLICE-2 polypeptide of the invention or an agonist thereof.
A still further aspect of the invention is related to a method for treating an individual in need of a decreased level of I-FLICE-1 or I-FLICE-2 activity in the body comprising, administering to such an individual a composition comprising a therapeutically effective amount of an I-FLICE-1 or I-FLICE-2 antagonist.